transfer and stability of the dehalogenase I gene of "Pseudomonas putida" PP3 by John Rutland Beeching

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Thesis (Ph.D.) - University of Warwick, 1984.

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Statementby John Rutland Beeching.
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Open LibraryOL14873058M

Download transfer and stability of the dehalogenase I gene of "Pseudomonas putida" PP3

An R-prime, pUU2, derived from the broad host-range plasmid R+ was generated carrying the dehalogenase I gene from Pseudomonas putida PP3.

This R-prime enabled its host to use 2-monoch1oropropionic acid (2MCPA) as sole carbon and energy source. The R-prime was studied by restriction endonuclease analysis. The process of R-prime formation was examined in detail and was Cited by: 2.

The transfer and stability of the dehalogenase I gene of. An R-prime, pUU2, derived from the broad host-range plasmid R+ was generated carrying the dehalogenase I gene from Pseudomonas putida PP3.

This R-prime enabled its host to use 2-monoch1oropropionic acid (2MCPA) as sole carbon and energy source. The R-prime was studied by restriction endonuclease : John Rutland Beeching. Dehalogenase genes of Pseudomonas putida PP3 on chromosomally located transposable elements Article (PDF Available) in Molecular Biology and Evolution 2(6) December with 38 Reads.

As a result of the production of two dehalogenases (DehI and DehII), Pseudomonas putida PP3 utilized halogenated alkanoic acids, such as 2-monochloropropionic acid (2MCPA), as sole sources of carbon and energy.

The DehI gene (dehI) was carried on a mobile genetic element (DEH) located on the chromosome of strain by: The α-haloacid dehalogenases have also been described for Pseudomonas putida strain PP3 with two different dehalogenating mechanisms described for its dehalogenases (DehI and DehII).

These dehalogenases catalyze substitution reactions at their chiral centres leading to the removal of halides from d -haloalkanoic acids and in some cases also the l -enantiomers [ 99 ]. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I -haloacid dehalogenase that can process both L- and D-substrates.

A 2-haloacid dehalogenase [6] enables the Gram-negative bacteria Xanthobacter autotrophicus GJ 10 and Pseudomonas putida PP3 to grow on chloroacetate or 2-chloropropionate. In both organisms, upstream of the dehalogenase structural gene, in the opposite direction of transcription, an open reading frame with homology to the family of adependent transcriptional activator proteins.

dehalogenase functions. The gene dehI was transcribed from a regulated promoter on DEHwhich was expressed andEscherichiacoli. Thedirection oftranscription ofdehIwasdetermined, andit was. The mobile genetic element,DEH found inPseudomonas putida PP3 carries a 2-haloalkanoic acid dehalogenase structural gene,dehI, and its associated regulatory gene,dehR I.

The. Beeching JR () The transfer and stability of the dehalogenase I gene of Pseudomonas putida PP3. PhD thesis, University of Warwick, Coventry, U.K. Google Scholar Beeching JR, Weightman AJ and Slater JH () The formation of an R-prime carrying the fraction I dehalogenase gene from Pseudomonas putida PP3 using the Inc P plasmid R DNA extraction, dehalogenase gene amplification, 16S rRNA gene amplification, and phylogenetic analysis.

DNA was extracted from bacterial biomass by the modified method of Ausubel et al. ().The modification used has been described elsewhere ().PCR amplification, cloning (40 clones were screened for each library), and sequencing of the deh genes from either the pure cultures or DNA from.

Gene regulation studies in pseudomonad bacteria are mainly restricted to Pseudomonas aeruginosa and Pseudomonas tutive promoters exhibit DNA sequences similar to the σ dependent constitutive promoters of Escherichia TOL meta-cleavage pathway operon promoter and the nah operon promoters are the best characterized σ dependent promoters, which exhibit regions rich.

The optimal temperature for DERA RE activity was 65 °C, but coupled with a rather low stability (half-life of 2 min). The highest stability was achieved at 25 °C. The new enzyme exhibits high resistance to organic solvents and acetaldehyde with a half-life being × higher compared to DERA EC under the exposure of mM acetaldehyde.

Hence it has the potential as a new promising biocatalyst with. The measured kinetic values for TOL transfer and stability can be used to predict the fate of TOL catabolic genes in a continuous suspended- growth reactor system consisting of P.

aeruginosa PAO and P. putida PB cells growing on a non-selective carbon-source. A.W., Slater, J.H. and Weightman, A.J. () The dehalogenase gene dehl. Batch mating experiments were employed to study the kinetics of the conjugal transfer of a TOL plasmid, using the transconjugant strain Pseudomonas aeruginosa PAO (TOL) as the plasmid donor and Pseudomonas putida PB and Pseudomonas aeruginosa PAO N as the plasmid recipients.

The genus Pseudomonas is the most heterogeneous and ecologically significant group of known bacte-ria. Some species of Pseudomonas are opportunistic pathogens that primarily cause nosocomial infec-tions (Brady et al., ). Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas.

The pgdS gene product shows the structural feature of a membrane-associated protein. We have constructed an overproduction system for dlhaloacid dehalogenase from Pseudomonas putida PP3 (dl. The crystallographic structure of the DehI homodimer is the first reported structure of a group I αHA dehalogenase (Fig.

1a).The monomer (Fig. 1b) is highly α-helical and is composed of a repeated motif, which is likely to have arisen from gene repeats (residues and ) share only 16% sequence identity yet can be superposed with an RMSD of Å ( out of. Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture.

Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4.

This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia. Enzymes in this class are unaffected by sulfhydryl-blocking reagents. The dehalogenase synthesized by Pseudomonas sp. dehalogenated both d- and l-2MCPA, although the l-isomer was dehalogenated more rapidly.

Pseudomonas putida PP3 was found to synthesize two 2HAA dehalogenases, designated Dehl and Dehll (22). The Pseudomonas Genome Database collaborates with an international panel of expert Pseudomonas researchers to provide high quality updates to the PAO1 genome annotation and make cutting edge genome analysis data available Search Gene Annotations by Name: Exact Name Name Contains Choose a specific strain (optional) OR choose a species (optional).

Pseudomonas putida KT is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P.

putida KT, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Horizontal transfer of the chlorothalonil hydrolytic dehalogenase gene (chd) is proposed based on the high conservation of the chd gene and its close association with a novel insertion sequence, ISOcsp1, in 16 isolated chlorothalonil-dechlorinating strains belonging to eight different ecological role of horizontal gene transfer is assumed to facilitate bacterial adaptation to.

Beeching, John Rutland () The transfer and stability of the dehalogenase I gene of 'Pseudomonas putida' PP3. PhD thesis, University of Warwick. Harrison, Lesley Ann () The characterisation of a cellulolytic microbial community isolated from soil.

PhD thesis, University of Warwick. Justin, Pauline M. () Physiology and biochemistry of the facultatively anaerobic. The sequences of DehD and Dhaloacid dehalogenase (HadD) from Pseudomonas putida AJ1 have 15% sequence similarity.

The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg, Arg and Tyr interacted with D-2CP. Thomas AW, Slater JH, Weightman AJ.

The dehalogenase gene dehI from Pseudomonas putida PP3 is carried on an unusual mobile genetic element designated DEH.

J Bacteriol. Mar; (6)– [PMC free article] Thomas AW, Topping AW, Slater JH, Weightman AJ. Topping AW, Thomas AW, Slater JH & Weightman AJ () The nucleotide sequence of a transposable haloalkanoic acid dehalogenase regulatory gene (dehRI) from Pseudomonas putida strain PP3 and its relationship with o dependent activators.

Biodegradation – CrossRef Google Scholar. Dehalogenase genes of Pseudomonas putida PP3 on chromosomally located transposable elements. Mol Biol Evol. Nov; 2 (6)– Smith JT. Wirkmechanismus der Chinolone. Infection. ; 14 (Suppl 1):S3– Stephens GM, Dalton H.

The effect of lipophilic weak acids on the segregational stability of TOL plasmids in Pseudomonas putida. Pseudomonas comprises a genus of species capable of utilizing a wide range of organic and inorganic compounds and of living under diverse environmental conditions. Consequently, they are ubiquitous in soil and water ecosystems and are important as plant, animal and human pathogens (Palleroni, ; Schroth et al., ).The genus Pseudomonas is well known for its metabolic versatility and.

A soil microcosm, containing a mixture of sand and a well-characterized phaeozem soil from loess, was developed for biodegradative applications. It was inoculated with soil-borneAlcaligenes xylosoxidans AB IV, degrading 2,2-dichloropropionate (DCPA), by a plasmid-encoded haloalkanoic acid halidohydrolase.

In long-term microcosm experiments, survival ofAlcaligenes xylosoxidans ABIV in the. The sequence identity between the Pseudomonas sp. Strain YL lhaloacid dehalogenase and the Xanthobacter autotrophicus GJ10 haloalkane dehalogenase is only 12%. 40 Furthermore, the recently reported crystal structure of the haloacid dehalogenase 41 shows that the two proteins are not structurally related.

The overall topology, as well as the. Phosphoryl transfer in Pseudomonas putida KTspecified PTS Ntr and organization of genes for PTS Ntr. (a) Phosphoryl transfer in PTS iations: PEP, phosphoenolpyruvate; and Pyr, pyruvate. (b) Gene organization for PTS Ntr components and mutation sites.

The genes with unknown functions are indicated by locus tag numbers. Cloning and expression of the MBA4 hdlIVa gene in Pseudomonas putida and in Escherichia coli suggested that the promoter of the Burkholderia gene is not regulated in these foreign hosts.

These results provide the basis for the hypothesis that genes normally silent in Burkholderia could be expressed constitutively, although at a basal level, in. Transfer of RP4-TOL from E. coli and P. putida to C. crescentus. WehaveusedtheRP4-TOLrecombinantplasmid as a model to study the transfer ofdegradative genes ofa Pseudomonas sp.

The plasmid was chosen because RP4 transfers at a very high frequency andexpresses well in C. crescentus (8). Moreover, RP4-TOL contains all the func.

Pseudomonas putida uses l-lysine as the sole carbon and nitrogen source which preferentially requires its metabolism through two parallel pathways. In one of the pathways δ-aminovalerate is the key metabolite, whereas in the other l-lysine is racemized to d-lysine, and l-pipecolate and α-aminoadipate are the key metabolites.

All the genes and enzymes involved in the d-lysine pathway, except. Dehalogenase genes of Pseudomonas putida Pp3 are located on mobile genetic elements, indicating the possibility of lateral transfer (Slater et al. ; Thomas et al. a,b; Weightman et al. Therefore, any further work to identify systems with the capacity to biodegrade HAAs will need to focus on the functional dehalogenase genes.

In Pseudomonas putida, genes for the glucose phosphorylative pathway and the Entner-Doudoroff pathway are organized in two operons; one made up of the zwf, pgl, and eda genes and another consisting of the edd, glk, gltR2, and gltS genes.

Divergently with respect to the edd gene is the gap-1 gene. Expression from P zwf, P edd,andP. Horizontal transfer of the chlorothalonil hydrolytic dehalogenase gene (chd) is proposed based on the high conservation of the chd gene and its close association with a novel insertion sequence, IS Ocsp1, in 16 isolated chlorothalonil-dechlorinating strains belonging to eight different genera.

The ecological role of horizontal gene transfer is assumed to facilitate bacterial adaptation to. Gene 32±39 halogenase gene by insertion element IS J Bacteriol Thomas AW, Slater JH, Weightman AJ (a) The dehalogenase ± gene dehI from Pseudomonas putida PP3 is carried on an un- Pritchard PH, Costa CF () EPA's Alaska oil spill bioremedi- usual mobile genetic element designated DEH.

J Bacteriol ation project. The gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR is a patchwork assembly of a Nag-like dioxygenase (dioxygenase belonging to the naphthalene dioxygenase NagAaAbAcAd family from Ralstonia sp.

strain U2) gene cluster and a chlorocatechol catabolism cluster. However, the transcriptional regulator gene usually present in the .Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy.

For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K production systems are convenient, yields of different fragments, even those produced from codon.Product # Description.

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